Method and diagnostic composition of detecting phenylketones



United States Patent Ofilice 3,048,475 Patented Aug. 7, 1962 spasmsMETHOD AND DIAGNUSTIC COMPOSHTIGN @F DETECTING PHENYLKETUNES Chauncey 0.Rape, Elkhart, Ind, assignor to Miles Laboratories, inc Ellrhart, Ind, acorporation of Indiana No Drawing. Filed June 16, 1958, Ser. No. 741,9936 Claims. (Ct. 23-430) This invention relates to a new and improveddiagnostic composition and to a method of testing for phenylketonuria.Particularly, this invention is concerned with a diagnostic compositionincorporated upon a bibulous strip which is useful for the qualitativedetection and quantitative estimation of phenylketones in body fluidsand especially phenylpyruvic acid in urine.

The presence of phenylpyruvic acid in urine is indicative ofphenylketonuria, a serious metabolic disorder resulting from theincomplete metabolism of aromatic amino acids. Pathologically,phenylketonuria manifests itself as a syndrome identified by severemental retardation, decreased pigmentation, epileptic seizures,dematoses and certain neurologic disorders, like poor motor control.Biochemically, phenylketonuria indicates the failure of the body tissuesto properly metabolize phenylalanine, which is an essential amino acidrequired for the synthesis of most proteins. In patients withphenylketonuria the excessive phenylalanine undergoes oxidativedeamination to phenylpyruvic acid (phenylpyruvate) which is excreted inthe urine.

One method of treating this disease is directed towards decreasing theintake of phenylalanine, since the mental retardation in phenylketonuriais due to intoxication by phenylalanine or one of its metabolites. Adiet low in phenylalanine is now commercially available and is incurrent use, viz. Ketonil, etc.

Since early detection and treatment of this biochemical aberration givespromise of normality, it is desirable that the urine of every baby oryoung child be tested on several occasions. Additional study isindicated in siblings of patients known to have phenylketonuria or inchildren in which there is the slightest suspicion of mentalretardation.

According to present practices in the art, urine specimens of suspectsare universally screened by means of a test involving several steps andutilizing a ferric chloride or a ferric ammonium sulfate solution. Theappearance within minutes of a deep green or blue-green color (sometimesan opaque deep blue-gray, owing to precipitated phosphates) afteraddition of a few drops of a 5% or 10% ferric chloride or a 10% ferricammonium sulfate solution to either the fresh urine of suspects or suchurine acidified by sulfuric acid or acetic acid may indicate thepresence of phenylpyruvic acid. This likelihood is then ordinarilyverified by subsequent reaction of such a urine specimen with a2,4-dinitrophenylhydrazine and isolation and identification of thehydrazone formed.

It has been found, however, that several substances interfere with thistesting procedure, such as bile, diacetic acid, salicylates, etc., andthat the color formed in consequence of such procedure is unstable,rather indistinct and prone to fade rapidly. It would be desirable,therefore, to have a simple test composition in convenient strip formpossessing qualities that would eifectively avoid the disadvantages justdiscussed.

Accordingly, it is an object of this invention to provide an improveddiagnostic composition for detecting phenylketonuria which reliablyindicates the presence of phenylpyruvic acid in urine and wherein thecolor is distinct and does not fade rapidly.

Another object of this invention is to provide a diagnostic compositionfor detecting phenylpyruvic acid in urine, which is in the form of abibulous strip or stick.

The advantages of a diagnostic in this form will become at once obvious:ease and simplicity of test procedure, absence of cumbersome equipment,ease of disposal of the test device, rapidity of testing procedure, etc.

Other objects as well as features and advantages of the invention willbecome apparent from the following detailed description:

A diagnostic composition according to the present invention comprises aniron salt as a source of ferric ions for the chromogenic reaction withphenylpyruvic acid, an organic acid to impede the hydrolysis of the ironsalt, and an additional salt as a phosphate complexing agent, all ofthese ingredients being carried by a bibulous base material or carrier,such as strips of filter paper. I prefer to prepare my diagnosticcomposition by dissolving these components in a suitable solvent ormixture of solvents and impregnating a bibulous body (e.g., a strip offilter paper) with the resulting solution, thereafter drying theimpregnated bibulous body.

Prior art, as indicated above, teaches the use of soluble ferricchloride or ferric ammonium sulfate in the presence of sulfuric acid fordetecting phenylpyruvic acid in urine. However, if such a solution wereused to impregnate a bibulous strip, no practical diagnostic productwould be obtained because of the instability of the ferric salt and thecorrosive action of sulfuric acid on the carrier. Strips made withferric chloride or ferric ammonium sulfate alone are likewise unstableand are not very sensitive.

It has been found that the addition of an organic acid, such as lactic,malic, succinic, cyclohexylsulfamic acids, greatly improves both thesensitivity and the stability of the strip. The organic acid in thecomposition reacts with ferric ions to form salts, such as ferriclactate, ferric malate, ferric succinate, etc., which are less-readilyhydrolyzed, thus checking the undesirable formation of ferric hydroxide.

It has also been found that the color reaction may be further improvedby addition of a phosphate complexing agent, such as magnesium sulfate,aluminum sulfate, aluminum ammonium sulfate, barium chloride, or calciumchloride. Without addition of such an agent, phosphates and otherconstituents of urine tend to bind ferric ions so that fewer areavailable for the chromogenic reaction with phenylpyruvic acid inconsequence of which the color indications are less distinct. Thepresence of Mg++, A1+++, Ba or Ca++, however, obviates this drawback.

Although the impregnating solution can be prepared by dissolving thecomponents in water only, it has further been found that an alcoholcontent of up to 20%, supplied by addition of ethanol, aids in producinga test strip which gives more uniform distribution of the reagentthroughout the impregnated area of the strip, and consequently moreuniform color indications.

N-propanol and isopropanol in concentrations of up to 10% as well asN-butanol in a concentration of up to 1% may be substituted for ethanolwith similar effects.

The invention will be illustrated, but is not limited, by the followingexamples:

EXAMPLE I Formulation of Preferred Impregnating Solution 3 vol. offerric ammonium sulfate, 0.4 F.W. (formula weight/ liter of solution).

3 vol. of magnesium sulfate, 1.2 M (molecular weight/liter of solution).

dissolving 19.3 g. of reagent grade ferric ammonium sulfate FeNHASOQ 12HO or Fe (SO (NHQ SO 241-1 in distilled water and diluting this to atotal volume of 100 ml. with distilled water.

Cyclohexylsulfamic acid solution was prepared by placing 21.5 g. inabout 75 ml. of water, gently heating until solution was complete andthen diluting it to a total volume of 100 ml. with distilled water.

Magnesium sulfate solution was prepared by dissolving 29.5 g. of reagentgrade magnesium sulfate (MgSO -7H O) in distilled water and diluting itto a total volume of 100 ml. with distilled water.

The impregnating solution was then prepared by mixing in 250 ml.Erlenmeyer flask at room temperature 30 ml. of the ferric ammoniumsulfate solution with 30 ml. of the magnesium sulfate solution, ml. ofthe cyclohexylsulfamic acid solution measured out while the solution waswarm (70 C.), since a 1.2 molar concentration cannot be obtained at roomtemperature (3.), and 20 ml. of special denatured ethyl alcohol, 2B.These solutions were thoroughly admixed by swirling the flask upon theaddition of each solution. The mixture thus prepared remains stablewithout any precipitation.

An equally operable impregnating solution may be prepared by weighingout solid chemicals and dissolving them in water or in a solutioncontaining 20 ml. of special denatured alcohol, 213, per 100 ml. ofsolution. Thus, 5.8 g. of ferric ammonium sulfate 8.9 g. of magnesiumsulfate (MgSO -7H O), 6.5 g. of cyclohexylsulfamic acid were dissolvedin water containing 20 ml. of special denatured alcohol, 2B, per 100 ml.of solution. Then the solution was diluted to a total volume of 100 ml.with the same alcohol-water solution.

The impregnating solution was poured into a shallow flat bottom dish toa depth of about inch.

Preparation of Test Strip Bibulous strips, such as filter paper cut intonarrow strips, small sticks of wood, or other porous or absorbingmaterial with a Water impervious barrier of ethyl cellulose /2 inch fromtip were dipped into the solution so that through the process ofsubmersion and capillary attraction the entire /2 inch of the strip upto the barrier was completely impregnated. The dipped strips were thenplaced in drying racks with the clipped ends up. The strips were allowedto stand on the laboratory desk at room temperature for 15 to 20 minutesand then placed in a hot air oven at 90110 C. for 15 minutes.

Other specific embodiments of my invention are illustrated by thefollowing examples:

EXAMPLE II Formulation 50 ml. of ferric ammonium sulfate, 0.4 F.W. 20ml. of gluconic acid, 1.2 M. 27.0 g. of aluminum ammonium sulfate. ml.of distilled water.

Preparation The ferric ammonium sulfate solution was prepared bydissolving 19.3 g. of reagent grade ferric ammonium sulfate [FeNH (SO-12H O OI Fe (SO 3 804 in distilled water and diluting it with distilledwater to a total volume of 100 ml. the gluconic acid, by dissolving 23.5g. of a-glucono-lactone in distilled water and diluting it withdistilled water to a total volume of 100 ml. Because of its limitedsolubility, aluminum ammonium sulfate [AlNH (SO --12H O, 27.0 g.] was iadded in solid form directly to the impregnating mixture along with 30ml. of distilled water. Heat was then gently applied till the solutionwas completed.

The impregnating mixture may also be produced by first mixing the solidcompounds and then dissolving them in water. Thus, 9.7 g. of ferricammonium sulfate, 27.0 g. of aluminum ammonium sulfate and 4.7 g. of6-glucono-lactone were weighed into a 250 ml. Erlenmeyer flask, about75-80 ml. of Watcr added, and the components dissolved by the gentleapplication of heat. V lhen the solution was complete, distilled waterwas added to a total volume of ml. The solution was kept at about 70 C.until the strips were dipped.

The test strips were then made as described in Example 1.

EXAMPLE III Formulation 50 ml. of ferric sulfate, 0.2 M 30 ml. ofmagnesium sulfate, 1.2 M 20 ml. of fi-glucono-lactone Preparation Theferric sulfate solution was prepared by dissolving 10 g. of reagentgrade Fe (SO -xI-I O in distilled water and diluting it with water to atotal volume of 100 ml.; the magnesium sulfate solution, by dissolving29.6 g. of reagent grade MgSO '7I-I O in distilled water and diluting itwith distilled water to a total volume of 100 ml.; and the gluconic acidsolution was prepared as described in Example Ii.

Again, the impregnating solution may be produced by first mixing thesolid compounds and then dissolving them in water. Thus, 5 g. of ferricsul- 8.9 g. of magnesium sulfate (MgSO -7H O) and 4.7 g. offi-glucono-lactone were weighed into a 250 ml. Erlenmeyer flask,dissolved in distilled water and diluted with distilled water to a totalvolume of 100 ml.

For the preparation of the test strips the procedure described inExample I was followed.

Procedure of Testing In use, an impregnated strip, made in accordancewith the invention, is dipped into the liquid specimen to be tested. Itwill give a positive reaction evidenced by a bluish-gray or green colorwithin 10 to 20 seconds when contacted with urine containing as littleas 100 ppm. of phenylpyruvic acid. Negative tests do not give thischaracteristic color but remain practically color-less or light butt incolor. The intensity of the color varies with the content ofphenylpymvic acid; from a light blue-gray at 100 ppm. to a darkbluish-gray at 3000 p.p.rn., depending on the composition of the teststrip. This phenomenon may be utilized for preparing a convenient colorchart.

In addition to the compositions described in the above examples, variousother combinations of ferric salts, or ganic acids, and salts ofmagnesium, aluminum, barium or calcium will give a satisfactory product.Chart 1 illustrates the possible combinations without, it is to beunderstood, limiting the scope of the invention to the compoundsenumerated.

CHART 2 Component Operable Range Optimum Content 1. Iron Salt 0.012FW/lUO 0.004 to 0.02 I'WV/lOO 0.008 to 0.05 moles/ 0.01 to 0.1 moles/1002. Organic Acid 3. Phosphate ComplexingAgent.

0.024 moles/100 0.05 moles/100 A satisfactory diagnostic composition,i.e., one that is stable and sensitive and gives a positive reactionwhen only 100 ppm. of phenylpyruvic acid are present in urine, may beobtained by regulating the ratio of the equivalents of ferric ions tothat of the organic acid to about 3 to 2 in the impregnating solution.This ratio of ferric salt to organic acid is tantamount to a solutionwith a pH of 1.0 to 1.8. For optimum testing results a pH of 1.5 isespecially preferred. If the pH is below 1.2, the test strip becomes toobrittle and fragile, and the color produced by a positive test fadesrapidly; if the pH is above 1.8, the composition turns brown and becomesinsensitive owing to the formation of ferric hydroxide and to thechelation of the ferric ions by the organic acid used.

In summary, this invention pertains to a diagnostic composition for thedetection of phenylketones in body fluids, especially phenylpyruvic acidin urine, which comprises a bibulous carrier or strip that has beenimpregnated With a composition consisting of an iron salt, an organicacid, and an additional salt which acts as a phosphate complexing agent,all of which are dissolved in a water-alcohol solution. The compositioncontaining ferric ammonium sulfate, cyclohexylsulfamic acid andmagnesium sulfate, constitutes the specially preferred embodiment ofthis invention. The ingredients of the composition are so adjusted as togive a pH of 1.0 to 1.8 of the impregnating solution; a pH of 1.5 isespecially preferred.

What is claimed is:

1. A diagnostic composition for the detection of phenylketones in bodyfluids which comprises a bibulous carrier impregnated with a solutionconsisting essentially of a source of ferric ions selected from thegroup consisting of ferric sulfate, ferric ammonium sulfate and ferricchloride, an organic acid as an iron stabilizing agent selected from thegroup consisting of cyclohexylsulfamic,

maleic, malonic, and succinic acids, and a phosphate complexing agentselected from the group consisting of magnesium sulfate, aluminumsulfate and aluminum ammonium sulfate, said solution having a pH Withina range of from 1.6 to 1.8.

2. A diagnostic composition for the detection of phenylketones in bodyfluids which comprises a bibulous carrier impregnated with a solutionconsisting essentially of ferric ammonium sulfate, cyclohexylsulfamicacid and magnesium sulfate, said constituents being so adjusted as toresult in a solution having a pH within a range of from 1.0 to 1.8.

3. A diagnostic composition for the detection of phenylketones in bodyfluids which comprises a bibulous carrier in strip form impregnated witha solution comprising from 1.9 to 9.7 wt. percent of ferric ammoniumsulfate, from 1.6 to 9.0 Wt. percent of cyclohexylsulfamic acid and from2.5 to 24.7 wt. percent of magnesium sulfate, said solution having a pHwithin a range of from 1.0 to 1.8.

4. A diagnostic composition for the detection of phenylpyruvic acid inurine which consists essentially of a bibulous strip which has beenimpregnated upon at least a portion thereof with a solution whichconsists essentially of about 5.8 wt. percent of ferric ammoniumsulfate, about 4.3 wt. percent of cyclohexylsulfamic acid and about 13.3wt. percent of magnesium sulfate, said solution having a pH of 1.8.

5. A diagnostic composition according to claim 3 wherein said pH is 1.5.

6. A method of detecting phenylketones in a body fluid which comprisesintroducing into said liquid a diagnostic composition consisting of abibulous carrier which has been impregnated upon at least a portionthereof with a solution which consists essentially of about 5.8 Wt.percent of ferric ammonium sulfate, about 4.3 Wt. percent ofcyclohexylsulfamic acid and about 13.3 wt. percent of magnesium sulfate,said solution having a pH of 1.8.

References Cited in the file of this patent UNITED STATES PATENTS2,577,978 Nicholls et a1. Dec. 11, 1951 2,848,308 Free Aug. 19, 19582,862,796 Gomberg Dec. 2, 1958 OTHER REFERENCES Fleury: Clinica ChimicaActa, vol. 2, 1957, pages 424 to 428.

Rupe et al.: Clin. Chem, 1957, vol. 3, pages 716727.

Hawk: Practical Phys. Chem, 13th ed., 1954, pages 840841.

Kenp: Biochem. Z., vol. 326, pages 14 to 17, 1954.

Nash et al.: Lancet, vol. 266, April-June 1954, pages 801-803.

Fiegl, Chem. of Spec., Select. and Sensitive Reaction, 1949, pages and91.

6. A METHOD OF DETECTING PHENYLKETONES IN A BODY FLUID WHICH COMPRISESINTRODUCING INTO SAID LIQUID A DIAGNOSTIC COMPOSITION CONSISTING OF ABIBULOUS CARRIER WHICH HAS BEEN IMPREGNATED UPON AT LEAST A PORTIONTHEREOF WITH A SOLUTION WHICH CONSISTS ESSENTIALLY OF ABOUT 5.8 WT.PERCENT OF FERRIC AMMONIUM SULFATE, ABOUT 4.3 WT. PERCENT OFCYCLOHEXYLSULFAMIC ACID AND ABOUT 13.3 WT. PERCENT OF MAGNESIUM SULFATE,SAID SOLUTION HAVING A PH OF 1.8.